The ability to analyze the chemical composition of the surface of a material with sub-micron spatial resolution is integral to a number of scientific disciplines. A variety of techniques are currently used for surface chemical analysis with sub-micron spatial resolution, including X-ray Photoelectron Spectroscopy (XPS) (Gaarenstroom (1993) Appl. Surf. 70:261); Secondary Ion Mass Spectroscopy (SIMS) (Chabala et al. (1995) Int. J. Mass. 143:191) Scanning Electron Microscopy (SEM) (Walther et al. (1992) J. Vilicrosc. O. 168:169); Auger Electron Spectroscopy (Prutton et al. (1995) Ultramicros. 59:47) and Friction Force Microscopy (FFM) (Overney (1995) Trends Poly. 3:359). All of these methods probe some specific property of the material being analyzed to determine the chemical composition. XPS, for example, measures the X-ray atomic photoelectron emission spectra of the sample being analyzed. FFM probes the local lateral interaction (friction) between the sample and an AFM tip as it is scanned across the sample with the friction being dependent on the chemical composition of both the tip and sample. The spatial resolution currently obtainable using these methods varies from 1.mu. for SIMS down to the Angstrom range for FFM. Unfortunately, the methods with the best spatial resolution, FFM and SEM, have poor chemical contrast.
Surface chemical analysis is also performed using spatially resolved mass spectrometry (MS). Spatially resolved mass spectrometry is currently conducted using an optically focused laser beam to desorb particles from the surface of the material being analyzed. (de Vries et al. (1992) Rev. Sci. Instrum. 63:3321-3325). Desorption is the process of removing particles from the surface of a material. The particles removed may be ions, atoms, molecules, clusters or larger structures. Once removed, the particles form a gas which is then analyzed to determine the composition of the material from which the particles were removed. Laser desorption generally refers to a technique in which photons provide the energy necessary to detach the particles from the surface of the material. Laser desorption is a "destructive analysis", in that, pieces of the material being studied are torn away during the analysis, thereby destroying the material.
Current laser desorption techniques can be briefly described with reference to FIG. 1. Referring to FIG. 1, a sample 5 has a surface 10 covered with particles 12. A light beam 15, such as a laser beam, is focused by optics 18 onto surface 10 to form an area of desorption 20. Particles 22 that are in the area of desorption 20 are detached from surface 10. An ionization beam (not shown) may pass above the surface 10 to ionize the desorbed particles 22.
In order to study the composition of a material by laser desorption, it is necessary that the light utilized be of the proper intensity and wavelength to remove the particles without destroying them. If the wavelength of the light is too short or the intensity is too high, particles 22 will likely undergo undesirable chemical reactions or be destroyed, rather than simply detach from the surface 10. On the other hand, if the wavelength of the light is too long or the intensity of the light is too weak, particles 22 will not detach from surface 10. The specific wavelength and intensity required will depend on the nature of the particles being removed. In general, light with a wavelength in the ultraviolet range is appropriate. The intensity of the light necessary is dependent upon the type of experiments being performed. For matrix-assisted laser desorption analysis (MALDI), an intensity of 10.sup.6 -10.sup.7 W/cm.sup.2 is necessary. Matrix-assisted laser desorption ionization is a method for producing ions in the gas phase. It is especially useful for studying large biological molecules. (Karas and Hillenkamp (1988) Anal. Chem 60:2299). The molecules of interest are suspended in a matrix, such as sinapinic or dihydroxybenzoic acid, and irradiated with a short duration laser pulse (approximately 3 nanoseconds), at a frequency 10-30 Hz. Upon irradiation the embedded analyte is ionized without decomposition, and the ions can then be analyzed by mass spectroscopy. For metal desorption an intensity of 10.sup.8 W/cm.sup.2 is necessary.
Although current laser desorption systems, such as those shown in FIG. 1, are able to provide the proper intensity and wavelength of light, it is difficult to achieve high spatial resolution with a laser. In this context, resolution refers to the accuracy in which the location of the particles being detached from the surface can be measured. As the resolution of the system is increased the determination of the location of the particles being detached from the surface becomes more accurate. In analyzing the composition of a subject material by laser desorption, it is important to be able to determine the location of particles being analyzed with a high degree of accuracy. Resolution is typically determined from the area of desorption (referred to as the spot size), which is the diameter of the laser beam on the surface. The smallest previously reported spot size is one micron, as discussed by deVries et al. (1992) Rev. Sci. Instrum. 63(6):3321-3325.
Two restraints--namely, diffraction and economic feasibility--limit the ability to achieve resolutions less than one micron in current laser desorption systems. Diffraction refers to the departure from rectilinear propagation of light waves that is experienced by light resulting from some obstruction of the wave front by an opaque surface. As can be seen in FIG. 2A, unobstructed light travels in straight lines, referred to as rectilinear propagation, if the light is passed through an aperture which is roughly equivalent to the wavelength of the light, however, propagation beyond the barrier is no longer strictly rectilinear, but rather, the light penetrates into regions beyond the barrier into regions that cannot be reached by a straight line drawn from the source. This phenomenon is called diffraction. From a theoretical standpoint, the resolving power of an optical focusing system is restricted by the diffraction limit 4.lambda.fo/.pi.d, where .lambda. is the wavelength, fo is the focal length of the lens and d is the beam width. To achieve high resolution with an optical focusing system high-precision lenses are required which are very expensive. Additionally, current laser desorption techniques require high precision optics to keep the laser narrowly collimated. These optics are expensive, and require time consuming precision alignment. Furthermore, different materials require different wavelengths to desorb. Therefore, when new materials are being studied the wavelength of the laser light must be changed. When the wavelength of the laser light is changed the optics must also be changed or resolution will be lost.
Spence et al. describes a more recent method for performing spatially resolved mass spectrometry in which the chemical composition of the surface is analyzed in one location at a time. Briefly, a scanning tunneling microscope (STM) tip is used to remove an atom (or group of atoms) from the sample being analyzed at a specific location. A high voltage pulse is then applied to the top to desorb and ionize this atom(s). The atom(s) is then guided to a Time-of Flight (TOF) detector to analyze its mass. The drawback of this method is that the system probes the sample in a single location only, leaves the surface to perform the mass spectral analysis and has to be returned to the surface with Angstrom reproducibility. This method leads to a number of technical problems and long analysis times, if surface mapping is the goal.
The methods of surface analysis currently available are not able to provide both high spatial resolution and the detailed chemical information that is desired. There is always a trade-off between resolution and chemical contrast, in that methods which provide high spatial resolution are unable to provide the detailed chemical information that is desired and methods which provide detailed chemical information lack the resolution that is desired. There remains a need, therefore, for a method of conducting surface analysis which provides both high spatial resolution and a detailed chemical analysis of the surface being studied.
Near-field scanning optical microscopy (NSOM) is a probe microscopy technique that was invented in 1972, as discussed by Ash et al. (1972) Nature 237:510. In NSOM a beam of light is passed through an aperture which is smaller than the wavelength of the light to optically and non-destructively image features on a surface. When substantially collimated light passes through an aperture which is smaller than the wavelength of the light, the light is spread out into what is referred to as a Fraunhofer diffraction pattern (see FIG. 2B). Referring to FIG. 2B, when substantially collimated light 35 strikes an opaque surface 40 and passes through an aperture 42, that is smaller than the wavelength of the light, a classical diffraction pattern 47 appears in the far-field 45, however, in the near-field 50, the light remains generally collimated to the size of aperture 42. Far-field 45 is generally the region more than one wavelength from the aperture. Near-field 50 is the region substantially less than one wavelength from aperture 42, and is approximately equal to the width of aperture 42. Thus, in the near field the illuminated area does not depend upon the wavelength of light, but rather depends only on the size of the aperture. NSOM uses this effect to perform optical microscopy with sub-wavelength resolution. The best reported spatial resolution using NSOM is approximately 20 nm.
The heart of any probe microscopy instrument lies in the shape of the tip of the probe. In NSOM, a sub-wavelength aperture must be constructed and scanned over the surface of the sample. Harootunian et al first developed tapered NSOM tips in 1986 by pulling quartz micro pipettes down to a point, followed by coating the outside of the pipettes with evaporated aluminum. (Harootunian et al (1986) Appl. Phys. Lett. 49:674). Betzig et al. have since found that quartz fiber optics serve this purpose even better. (Betzig et al. (1991) Science 251:1468). The fiber optics have a natural degree of collimation along the propagating axis, resulting in a larger amount of light reaching the end of the tip, which enhances the intensity for smaller and smaller aperture sizes.
It is an object of the present invention to provide a laser desorption technique and apparatus which provides high spatial resolution together with a detailed chemical analysis of the surface being analyzed.
Another object of the present invention is to provide a laser desorption technique and apparatus that is easy and inexpensive to construct and operate.
A further object of the present invention is to provide a stable laser beam for laser desorption.
Yet another object of the present invention is to provide a laser desorption technique and apparatus which desorbs in a spot with a size which is wavelength independent.
Even another object of the present invention is to utilize methods from near-field scanning optical microscopy and time-of-flight mass spectrometry to achieve the above objects.
Additional objects and advantages of the invention will be set forth in the description which follows and in part will be obvious from the description, or may be learned by the particulars of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the claims.